Sara Soth
AM BioTech
10/29/2013
Mystery Plasmid Lab Write up
Objective
To determine which plasmids are which by doing a mini-prep to obtain the purified plasmids.
Use purified plasmids to run a gel and verify each plasmid by comparing them to the virtual
prediction through NEB Cutter. Alternative is to grow with a bacteria on an Agar Arabinose
Ampicillin petri dish.
Background Information
When three plasmids ( pAmp, pKan and pGlo) were ordered from Doofenshmirtz’s company,
the samples arrived unlabeled. Using the mini-prep kit – including a column microfuge reaction
tube - the plasmids were separated from the media they came in.
Once the media and the bacteria with the plasmids were separated, many spin downs were
required to purify the plasmids and separate them from the bacteria themselves. The samples
were spun down in this order: Buffer P1, Buffer P2, Buffer N3, Buffer PB, and then Buffer PE.
Experimental Design
A mini-prep will be used to extract the bacteria from the broth and then to extract the plasmids
from the bacteria. Once plasmids are purified and the last of the ethyl alcohol has evaporated
(which will leave the sample in a small dried pellet at the bottom of a reaction tube. Once it is
visible that the alcohol is gone, the plasmid DNA will be rehydrated with Buffer EB.
Once the purified plasmid DNA is obtained, the DNA will be recombined with a weak strain of
E.coli and will be grown on an Agar Arabinose Ampicillin petri dish. It is predicted that pGlo will
glow when it reacts with the petri dish gel, pAmp will survive because it has built up immunity
to Ampicillin (so does pGlo), and pKan will die as it does not have the trait for Ampicillin
resistance.
Materials

Mini Prep Kit (QIAprep spin column)

Buffer 1

Buffer 2

Buffer PB

Buffer PE

Buffer EB

Buffer N3

Microcentrifuge

Arabinose Ampicillin Agar petri dish

Mystery Plasmids

Buffer for Plasmids

Micropipettes and corresponding tips

NanoDrop

Beaker to discard tips in
Method
1. Resuspend pelleted bacteria cells in 250 uL Buffer P! and transfer to a microcentrifuge
tube.
2. Add 250uL of Buffer P2 and gently invert tube 4-6 times to mix.
3. Add 350uL Buffer N3 and invert the tube immediately but gently 4-6 times.
4. Centrifuge for 10 minutes at 13,000 rpm in a table top micro centrifuge.
5. Apply the supernatant from step 4 to the QIA spin column by decanting or pipetting.
6. Centrifuge for 30-60 seconds. Discard the flow-through.
7. Wash the spin colum by adding 0.5 mL Buffer PB and centrifuging for 30-60 seconds.
Discard the flow through.
8. Wash spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds.
Discard the flow through and centrifuge for an additions 1 minute to remove residual
wash buffer.
9. Place the spin column in a clean 1.5 mL microcentrifuge tube. To elute the DNA, add
50uL Buffer EB or water to the center of each spin column, let stand for 1 minute and
then centrifuge for 1 minute.
10. Grow each plasmid in bacteria on Arabinose Agar petri dishes (1 plate per bacteria).
11. Take colony from each sample and grow on Arabinose Ampicillin Agar petri dish.
12. View under UV light.
Results
From the picture above, it can be concluded that sample one was pGlo because it is glowing,
sample two was pAmp because it survived, but is not glowing, and sample three is pKan
because it died.
Conclusion
This lab actually went very well. The results are concise and easy to decipher. The objective of
this lab has been achieved since it is now possible to tell which plasmid is which by looking at
the samples in the petri dish. The samples that survived are the samples that have Ampicillin
resistance coded into their DNA. Since pKan died because it didn’t have resistance it is now
known that the other two samples are pAmp and pGlo. Knowing that pGlo will glow when it
reacts with the petri dish, sample number one is ruled out as being pGlo. Due to the process of
elimination, sample number three must be pAmp – alive, but not glowing.
Although this lab went well, it is possible that there is a certain level of contamination. By
looking at the picture in the above Results section, a ring of glowing bacteria circles the whole
petri dish. Knowing this, it is possible that a drop of a contaminant fluid got into the petri dish
(though it is most likely that it is just condensed water from making the gel.